Elsevier

Experimental Eye Research

Volume 89, Issue 6, December 2009, Pages 960-966
Experimental Eye Research

Quantitative and regional measurement of retinal blood flow in rats using N-isopropyl-p-[14C]-iodoamphetamine ([14C]-IMP)

https://doi.org/10.1016/j.exer.2009.08.005Get rights and content

Abstract

Quantitative and regional measurement of retinal blood flow in rodents is of prime interest for the investigation of regulatory mechanisms of ocular circulation in physiological and pathological conditions. In this study, a quantitative autoradiographic method using N-isopropyl-p-14C-iodoamphetamine ([14C]-IMP), a diffusible radioactive tracer, was evaluated for its ability to detect changes in retinal blood perfusion during hypercapnia. Findings were compared to cerebral blood flow values measured simultaneously. Hypercapnia was induced in awaken Wistar rats by inhalation of 5% or 8% CO2 in medical air for 5 min. [14C]-IMP (100 μCi/kg) was injected in the femoral vein over a 30 s period and the rats were sacrificed 2 min later. Blood flow was calculated from whole-mount retinae and 20 μm thick brain sections in discrete regions of interest by quantitative autoradiography or from digested samples of retina and brain by liquid scintillation counting. Retinal blood flow values measured with quantitative and regional autoradiography were higher in the central (108 ± 20 ml/100 g/min) than in peripheral (84 ± 15 ml/100 g/min) retina. These values were within the same range as cortical blood flow values (97 ± 4 ml/100 g/min). The retinal blood flow values obtained on whole-mount retinae were validated by the sampling method. Hypercapnia significantly increased overall blood flow in the retina (24–53%) with a maximal augmentation in the peripheral region and in the brain (22–142%). The changes were stronger in the brain compared to retina (p = 0.016). These results demonstrate that retinal blood flow can be quantified using [14C]-IMP and compared with cerebral blood flow. This technique is a powerful tool to study how retinal blood flow is regulated in different regions of the rat retina.

Introduction

Deficit in blood supply in the retina contributes to the development of retinal diseases, such as diabetic retinopathy, age-related macular degeneration and glaucoma (Grunwald et al., 1984, Langham et al., 1991, Atmaca et al., 1996). Rodent models are commonly used to study the development of these retinal damages and to test potential therapies. However, blood flow dysfunctions in rodents and their cellular and molecular mechanisms remain elusive. This is principally due to a lack of a technical approach which would adequately assess all the specific features of the rodent retinal microcirculation.

Due to its small size, laminar structure, location and apposition to the choroid, the rodent retina is particularly difficult to study. Optically based imaging techniques, including laser Doppler flowmetry (Tsujikawa et al., 2000, Yu et al., 2005, Chauhan et al., 2006, Mori et al., 2007), on-line video angiography (Clermont et al., 1994, Kunisaki et al., 1998) or optical coherence tomography (Fujimoto et al., 1995) have a great temporal resolution but a poor spatial and laminar resolution and require a high transparency of the light paths (Glazer, 1988, Duong et al., 2008). Alternatively, quantitative techniques such as the use of systemic radioactive or non-radioactive microspheres (Chemtob et al., 1991, Alm et al., 1997, Wang et al., 2007) and magnetic resonance imaging (Sicard and Duong, 2005, Duong et al., 2008, Li et al., 2008) are performed on anesthetised animal which modifies cardiovascular parameters. The use of radioactive diffusible tracer iodoantipyrine commonly used for the measurement of cerebral blood flow (CBF) (Sakurada et al., 1978, Bryan et al., 1988, Vaucher et al., 1997, Greenberg et al., 1999) has also been tested for quantitative measurement of retinal blood flow by autoradiography in the cat, monkey (Sossi and Anderson, 1983, Quigley et al., 1985) and rat (O'Brien et al., 1997), but post-mortem tracer diffusion limits its spatial resolution (Caprioli and Miller, 1988).

In order to simultaneously provide quantitative and local measurement of discrete changes of the retinal blood flow (RBF) in conscious rats, the autoradiographic technique was adapted in the present study by using the radioactive diffusible tracer N-Isopropyl-p-[14C]-iodoamphetamine ([14C]-IMP). This tracer was chosen because it has an insignificant post-mortem diffusion so that tissue perfusion could be quantified in whole-mount retina isolated from the choroid. The principle of blood flow evaluation with [14C]-IMP is identical to that of micrometric microspheres (Wang et al., 2007) except that this “molecular microsphere” –IMP– is freely diffusible (does not depend on the blood-retinal or blood–brain barrier) and is trapped within the tissue instead of the microvascular space. It is then possible to quantify tissue perfusion at a microvascular level throughout the retina by computerized autoradiography. Moreover, the use of a diffusible tracer avoids inaccuracies of the RBF measurement due to axial streaming, plugging or permeability changes as reported with the classical microsphere technique (Glazer, 1988, Wang et al., 2007). Autoradiography technique is usually performed on 20 μm thick brain sections, whereas the flat mount retina is on average 200 μm thick. In order to validate RBF values measured by autoradiography, samples of the retina were digested and analyzed by liquid scintillation counting (O'Brien et al., 1997). Finally, as hypercapnia is commonly used to test the metabolic vasodilatation, two hypercapnia regimens were used to challenge the sensitivity of this technique to assess RBF changes. In addition, this technique provided the opportunity to simultaneously assess the RBF and CBF, which is an additional advantage to assess the tissue specificity of the responses to diverse physiological stresses.

Section snippets

Animals

Male Wistar rats (n = 20; 200–250 g) purchased from Charles River (St-Constant, Qc, Canada) were used for the measurement of RBF and CBF using [14C]-IMP by autoradiography or the sampling method. Rats were housed individually and placed in a room at 23 °C with a 12 h light/dark adapted photoperiod, with food and water provided ad libitum. All experimental methods and animal care procedures were approved by the local institutional animal care committee, “Comité de Déontologie de

Quantitative and regional measurement of retinal blood flow in control animals

Pseudocolor autoradiograms of whole-mount retinae displayed a gradient of blood flow from the center of the retina to the periphery (Fig. 2, Table 1). The blood flow values were significantly higher in the optic nerve head region (108 ± 20 ml/100 g/min) and isopter 1 mm (112–120 ml/100 g/min, according to the quadrants examined) than in the isopter 3 mm (82–86 ml/100 g/min, according to the quadrants examined, p < 0.05, Table 1). The values of blood flow throughout each concentric isopter were

Discussion

This study used an autoradiographic method for the simultaneous quantification of RBF and CBF in non-anesthetised rats. The use of the diffusible tracer [14C]-IMP provided quantitative measurements of RBF in discrete regions of the retina ranging from the central to peripheral retina. This technique was sensitive enough to quantify differential RBF changes related to two different hypercapnia regimens. Moreover, it allowed for the comparison of blood flow values of the retina to those of the

Conclusion

In summary, we demonstrated that RBF can be quantitatively and regionally measured with [14C]-IMP in rats. This offers a beneficial opportunity for ophthalmology research since most ocular diseases are characterized by discrete and circumscribed changes in blood flow. The possibility to compare quantitative data of RBF in different groups is essential for the evaluation of pharmaceutical treatments or to detect the subtle changes of blood flow in the retina of ocular diseases models.

Acknowledgements

The authors would like to thank Florence Dotigny for her technical assistance, Denis Latendresse for his graphical help, Mark Burke and Pierre Lacombe (Université Paris VII) for careful reading of the manuscript and helpful discussion. This study was supported by CNIB Baker New Researcher Fund, Canadian Foundation for Innovation (E.V.) and the Vision Research Network (FRSQ). MP is a recipient of the graduate student scholarship from the Foundation Fighting Blindness. EV received a

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